Trinucleotide Repeat Instability via DNA Damage and Repair

描述(申请人提供)：遗传性三核苷酸重复序列(TNR)不稳定(即扩张和缺失/收缩)与40多种人类家族性神经退行性疾病和癌症有关。非遗传性躯体TNR不稳定可能参与了这些疾病在普通公众中的发生发展。目前还没有有效的治疗TNR相关疾病的方法，部分原因是对潜在机制的了解不足。我们最近发现，DNA碱基损伤和碱基切除修复(BER)通过诱导单链DNA(SsDNA)断裂和促进GC自碱基配对发夹的形成来启动和调控体细胞CAG重复序列的扩张和缺失。这表明DNA碱基损伤、单链DNA断裂和BER在调节TNR不稳定性中发挥了新的作用。为了探索DNA损伤和BER作为预防和治疗TNR相关疾病的新靶点的潜力，在本项目中，我们试图了解环境和化疗诱导的单链DNA断裂及其无效修复是如何参与BER过程中体细胞TNR不稳定的。这一目标将通过追求三个具体目标来实现。目的1是确定环境和化疗导致的DNA碱基损伤和单链DNA断裂的积累是否可以以一种特定的方式优先导致CAG重复序列不稳定。将确定由环境毒物和化疗药物如氯乙烯和替莫唑胺诱导的单链DNA断裂在CAG/CTG重复区段中的定点积累。DNA损伤剂诱导的单链DNA断裂积累的独特模式将与重复序列的扩张和缺失相关联，以确定损伤特异性的CAG重复序列不稳定的“位置效应”。在误码率酶和辅因子水平不平衡的情况下，将进一步研究这些影响，以确定TNR的不稳定性是否可以通过降低误码率效率来调节。目的2验证低效误码率通过促进多种非B-型DNA结构的形成而促进CAG重复缺失的假说。这将通过确定脱氧核糖核酸聚合酶(POLçGenetic Variants，POLκ)的低效DNA合成是否有助于模板发夹的积累和促进TnR缺失来实现。目的3是确定是否可以通过BER蛋白质-蛋白质相互作用和功能协调有效地破坏非B-型DNA结构来防止TNR的扩张和缺失。本项目通过剖析环境和化疗DNA损伤、BER和TNR不稳定性之间的相互作用，研究了DNA损伤诱导体细胞TNR不稳定性的基本机制。这些结果将为以下方面提供重要的新见解：暴露在环境和化疗压力下可能如何影响普通人群中TNR相关人类疾病的发展和进展，以及如何通过DNA损伤修复来防止这些不利影响。这将有助于确定预防、诊断和治疗TNR相关疾病的新靶点，并为环境和化疗引起的遗传毒性效应的风险评估提供新的信息。

DESCRIPTION (provided by applicant): Inherited trinucleotide repeat (TNR) instability, (i.e. expansions and deletions/contractions) is associated with more than 40 human familial neurodegenerative diseases and cancer. Non-inherited somatic TNR instability may be involved in the development of these diseases in the general public. No effective treatment for TNR- related diseases is yet available, partially because of a poor understanding of the underlying mechanisms. We have recently discovered that DNA base damage and base excision repair (BER) initiate and modulate somatic CAG repeat expansion and deletion by inducing single-strand DNA (ssDNA) breaks and promoting the formation of GC self-base-pairing hairpins. This indicates a new role of DNA base lesions, ssDNA breaks and BER in modulating TNR instability. To explore the potential of DNA damage and BER as new targets for the prevention and treatment of TNR-related diseases, in this project we seek to understand how environmentally and chemotherapeutically induced ssDNA breaks and their inefficient repair are involved in somatic TNR instability during BER. This goal will be achieved by pursuing three Specific Aims. Aim 1 is to determine if the accumulation of environmentally and chemotherapeutically induced DNA base lesions and ssDNA breaks can preferentially lead to CAG repeat instability in a site-specific manner. Site-specific accumulation of the ssDNA breaks in CAG/CTG repeat tracts induced by environmental toxicants and chemotherapeutic agents such as vinyl chloride and temozolomide will be determined. The unique patterns of ssDNA break accumulation induced by DNA-damaging agents will be correlated with repeat expansion and deletion to identify damage- specific "position effects" on CAG repeat instability. The effects will be further examined under imbalanced levels of BER enzymes and cofactors to determine if TNR instability can be modulated by compromised BER efficiency. Aim 2 is to test the hypothesis that inefficient BER facilitates CAG repeat deletion by promoting the formation of multiple non-B-form DNA structures. This will be done by determining if inefficient DNA synthesis by DNA polymerases (Pol ß genetic variants, Pol κ) can facilitate the accumulation of a template hairpin and promote TNR deletion. Aim 3 is to determine if TNR expansion and deletion can be prevented by efficiently disrupting non-B-form DNA structures through BER protein-protein interactions and functional coordination. This project addresses the fundamental mechanisms underlying DNA damage-induced somatic TNR instability by dissecting the interplay among environmental and chemotherapeutic DNA damage, BER, and TNR instability. The results will provide important new insights into how exposure to environmental and chemotherapeutic stresses may influence the development and progression of TNR-related human diseases in the general population, and how these adverse effects can be prevented by DNA damage repair. This will help to identify novel targets for prevention, diagnosis, and treatment of TNR-related diseases, and provide new information for risk assessment of environmentally and chemotherapeutically induced genotoxic effects.